Oligonucleotide compositions and their use to induce differentiation of cells

ABSTRACT

The present invention provides compositions comprising a 3′-OH, 5′-OH, chemically unmodified, synthetic phosphodiester nucleotide sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO: 4, and a pharmaceutically acceptable carrier, wherein the compositions are useful to induce differentiation of cells or to stimulate differentiation or production of pluripotent cells. The present invention provides methods of using these compositions to induce differentiation of pluripotent cells, including bone marrow derived cells, and to treat disease associated with insufficient differentiation of cells in animals and humans, including but not limited to leukemia, lymphoma, non-malignant blood disorders such as hemoglobinopathies, sickle cell disease, myelodysplastic syndrome, pancytopenia, anemia, thrombocytopenia and leukopenia.

PRIOR RELATED APPLICATIONS

[0001] The present application claims priority to U.S. provisionalpatent application serial No. 60/286,158 filed Apr. 24, 2001.

FIELD OF THE INVENTION

[0002] The present invention provides compositions comprising specificoligonucleotides combined with a pharmaceutically acceptable carrier,wherein the compositions are useful to induce differentiation of cells,including pluripotent cells, leukemic cells, lymphoma cells and bonemarrow-derived cells, and to treat diseases such as leukemia, lymphomaand disorders associated with insufficient differentiation of cells.

BACKGROUND OF THE INVENTION

[0003] Numerous diseases and conditions in animals and humans areassociated with insufficient differentiation of cells or with aninsufficiency of cells. Many of these cells are derived from bonemarrow. Such diseases and conditions include but are not limited toleukemia, lymphoma, and non-malignant blood disorders such ashemoglobinopathies, sickle cell disease, myelodysplastic syndrome andinsufficient production of bone marrow derived cells following therapiessuch as radiation and chemotherapy.

[0004] Differentiation therapy of leukemia cells in diseases such asacute promyelocytic leukemia (APL), acute myeloid leukemia (AML),chronic promyelocytic leukemia (CPL) and chronic myeloid leukemia (CML),has provided an alternative strategy for the treatment of leukemia. Indifferentiation therapy, immature leukemia cells are induced bydifferent chemical compounds to attain a mature phenotype resulting inarrest of their growth.

[0005] A number of differentiation compounds and also radiation havebeen reported to induce the differentiation of leukemia cells. Hemin,butyric acid, 5-azacytidine, cytosine arabinoside, hydroxyurea,guanosine, guanine, retinoic acid, trimidox, gamma-irradiation,mithramycin and chromomycin have been reported to induce differentiationof leukemia cells (Rutherford et al., Nature 280:164, 1979; Gambari etal., Biochem. Biophys. Acta, 886:203, 1986; Bianchi et al., Cancer Res.46:6327, 1986; Adunyah et al., Biochem. Biophys. Acta, 1263:123, 1995;Osti et al., Haematologica 82:395, 1997; Cortesi et al., Eur. J.Haematol. 61:295, 1998; Iyamu et al., Biochem. Biophys. Res. Com.247:759, 1998; Schwenke et al., Leuk. Res. 19:955, 1995; and Bianchi etal., Br. J. Haematol. 104:258, 1999).

[0006] Synthetic oligonucleotides are polyanionic sequences that areinternalized in cells (Vlassov et al. Biochim. Biophys. Acta 1197:95,1994). Synthetic oligonucleotides are reported to bind selectively tonucleic acids (Wagner, R. Nature: 372:333, 1994), to specific cellularproteins (Bates et al. J. Biol. Chem. 274:26369, 1999) and to specificnuclear proteins (Scaggiante et al. Eur. J. Biochem. 252:207, 1998), andto inhibit proliferation of cancer cells. Synthetic oligonucleotideshave not been reported to possess differentiating activity on acuteand/or chronic pro-myelocytic cells and/or myeloid leukemia cells.Synthetic phosphorothioate oligonucleotides having a CpG motif(5′purine-purine-cytosine (C)-guanine (G)-pyrimidine-pyrimidine3′) havebeen shown to induce the proliferation of B-cell chronic lymphocyticleukemia (Decker et al., Blood 95:999, 2000). Synthetic 27 basesequences containing G and variable amounts of thymine (T), hereinafteroligonucleotide GTn, wherein n is ≧1 or ≦7 Ts (Scaggiante et al., Eur.J. Biochem. 252:207, 1998), and wherein the number of bases is >20(Morassutti et al., Nucleosides and Nucleotides 18:1711, 1999), havebeen reported to inhibit growth of leukemia cells by sequence specificbinding to a 45 kDa nuclear protein. In contrast, GTn sequences, whereinthe total number of bases is less than 15, are reported to be inactiveagainst these cells (Morassutti et al. Nucleosides and Nucleotides18:1711, 1999). Chimeric methylphosphonodiester/phosphodiesteroligonucleotides of sequence type CGNNN (N=A, C, G or T), introducedinto the cytoplasm of cells by 10 minutes of reversible permeabilizationwith streptolysin O, induce apoptosis of human T cell leukemia cells.Nevertheless, the CGNNN oligonucleotides are reported to be inactiveagainst three CML cell lines (K562, LAMA84 and KYO1), showing nosignificant effect on the growth and survival of these cells (Tidd etal., Nucleic Acid Res. 28:2242, 2000).

[0007] Depletion of bone marrow derived cells is observed in severalconditions, including depletion following radiation therapy orchemotherapy. Insufficient production of cells destined to becomeerythrocytes or granulocytes is associated with numerous problems,including but not limited to, reduced delivery of oxygen to cells,decreased immune function, and clotting abnormalities. Varioustherapies, including expensive chemotherapies, are often required tostimulate production of red and white cells.

[0008] Most prior art differentiating therapies have proven to be lessthan adequate for clinical applications. Many of these therapies areinefficient or toxic, have significant adverse effects and aredebilitating for the recipient. Therefore, there is a continuing needfor novel compositions and methods that induce differentiation of cellssuch as myeloid-derived leukemia cells. What is also needed are newtherapeutic compositions and methods that stimulate production anddifferentiation of pluripotent cells such as bone-marrow derived cells.Also needed are new therapeutic compositions that induce differentiationof cells. What is also needed are compositions and methods that may beused to treat diseases and conditions characterized by insufficientdifferentiation of cells or insufficient production of marrow derivedcells.

SUMMARY OF THE INVENTION

[0009] The present invention fulfills these needs by providing a methodcomprising administration of a composition comprising a 3′-OH, 5′-OH,chemically unmodified, synthetic phosphodiester oligonucleotide sequence(hereinafter sequence) selected from the group consisting of SEQ ID NO:1 (5′GTG3′), SEQ ID NO: 2 (5′GGGTGG3′), SEQ ID NO: 3 (5′GGGAGG3′) andSEQ ID NO: 4 (5′CCACCC3′) and a pharmaceutically acceptable carrier,wherein the composition induces differentiation of cells. The presentinvention provides a method to treat diseases associated with growth ofcells that are not differentiated to a mature phenotype. Terminaldifferentiation of cells may include one or more responses selected fromthe group consisting of induction of erythrocyte-like phenotype,monocyte-like phenotype, megakaryocyte-like phenotype, inhibition ofproliferation of leukemia cells and induction of hemoglobin synthesis.

[0010] The compositions of the present invention may be used to treatdiseases related to insufficient differentiation of cells. Such diseasesinclude but are not limited to leukemia, lymphoma, and non-malignantblood disorders such as hemoglobinopathies, sickle cell disease ormyelodysplastic syndrome. The compositions of the present invention arebelieved to be useful for treatment of pancytopenia, anemia,thrombocytopenia and leukopenia. Other conditions that may be treatedwith the compositions of the present invention include lymphoma andnonmalignant blood disorders, including but not limited tohemoglobinopathies, sickle cell disease and myelodysplastic syndromes.In a preferred embodiment, the compositions of the present invention areadministered to an animal or a human with leukemia in an amounteffective to treat the leukemia.

[0011] The compositions of the present invention may also beadministered to an animal or human together with other therapies as acombination therapy. These therapies may include administration oftherapeutic compounds or radiation therapy. The compositions of thepresent invention may be administered before, after, or concomitantlywith the other therapy. Such combination therapy may augment the nettherapeutic effect on the animal or human. The compositions of thepresent invention may be administered alone, or in combination withother therapeutic modalities including, but not limited to,chemotherapeutic agents, differentiating agents, immunotherapeuticagents, antimicrobial agents, antiviral agents or in combination withradiation therapy.

[0012] The compositions of the present invention may be administered toa recipient to stimulate production of cells after other therapiesadministered to the recipient have depleted such cells. One non-limitingexample involves depletion of cells derived from bone marrow followingradiation therapy or chemotherapy. The compositions of the presentinvention may also be administered to a recipient to stimulateproduction and differentiation of other cells such as pluripotent stemcells, myeloid stem cells, lymphoid stem cells, progenitor cells, immunecell precursors, and/or other cells derived from these pluripotent stemcells, myeloid stem cells, lymphoid stem cells, progenitor cells, andimmune cell precursors. The compositions of the present invention mayalso be administered to a recipient to stimulate production anddifferentiation of cells from numerous sources, including but notlimited to, bone marrow, liver, spleen, lymph nodes, thymus and cordblood.

[0013] The compositions of the present invention may also beadministered in vitro to affect differentiation of cells such aspluripotent stem cells, myeloid stem cells, lymphoid stem cells,progenitor cells, immune cell precursors, and/or other cells derivedfrom these pluripotent stem cells, myeloid stem cells, lymphoid stemcells, progenitor cells, and immune cell precursors.

[0014] The unexpected and surprising ability of the composition of thepresent invention to induce differentiation of bone-marrow derivedcells, including leukemia cells, addresses a long-felt, unfulfilled needin the medical arts and provides an important benefit for animals andhumans.

[0015] Accordingly, it is an object of the present invention to providea method comprising administration of a composition comprising a 3′-OH,5′-OH, chemically unmodified, synthetic phosphodiester oligonucleotidesequence (hereinafter sequence) selected from the group consisting ofSEQ ID NO: 1 (5′GTG3′), SEQ ID NO: 2 (5′GGGTGG3′), SEQ ID NO: 3(5′GGGAGG3′) and SEQ ID NO: 4 (5′CCACCC3′) and a pharmaceuticallyacceptable carrier to treat disease in animals and humans, wherein thedisease is characterized by insufficient differentiation of cells.

[0016] Another object of the present invention is to provide a methodcomprising administration of a composition comprising a 3′-OH, 5′-OH,chemically unmodified, synthetic phosphodiester oligonucleotide sequence(hereinafter sequence) selected from the group consisting of SEQ ID NO:1 (5′GTG3′), SEQ ID NO: 2 (5′GGGTGG3′), SEQ ID NO: 3 (5′GGGAGG3′) andSEQ ID NO: 4 (5′CCACCC3′) and a pharmaceutically acceptable carrier toinduce progenitor cell maturation and differentiation in animals andhumans.

[0017] Another object of the present invention is to provide acomposition and method to treat leukemia.

[0018] Yet another object of the present invention is to provide amethod that inhibits proliferation of leukemic cells and inducesdifferentiation of leukemic cells.

[0019] Another object of the present invention is to provide a method totreat lymphoma.

[0020] Yet another object of the present invention is to provide amethod to treat non-malignant blood disorders.

[0021] Still another object of the present invention is to provide amethod to treat hemoglobinopathies.

[0022] A further object of the present invention is to provide a methodto treat sickle cell disease.

[0023] Yet another object of the present invention is to provide amethod to treat myelodysplastic syndrome.

[0024] Another object of the present invention is to provide a method totreat pancytopenia.

[0025] Yet another object of the present invention is to provide amethod to treat anemia.

[0026] A further object of the present invention is to provide a methodto treat thrombocytopenia.

[0027] Another object of the present invention is to provide a method totreat leukopenia.

[0028] Still another object of the present invention is to provide amethod to induce progenitor cell maturation and differentiation.

[0029] Yet another object of the present invention is to provide amethod to induce maturation and differentiation of cells including butnot limited to pluripotent stem cells, myeloid stem cells, lymphoid stemcells, progenitor cells, immune cell precursors, and/or other cellsderived from these pluripotent stem cells, myeloid stem cells, lymphoidstem cells, progenitor cells, and immune cell precursors.

[0030] Still another object of the present invention is to provide amethod to induce bone marrow-derived progenitor cell maturation.

[0031] Another object of the present invention is to provide a methodthat increases the number of bone marrow derived-cells followingtreatment with therapeutic agents.

[0032] Yet another object of the present invention is to provide amethod that increases the number of bone marrow derived-cells followingtreatment with chemotherapeutic agents.

[0033] Another object of the present invention is to provide a methodthat restores the number of bone marrow derived-cells followingtreatment with radiotherapy.

[0034] Still another object of the present invention is to provide amethod that restores the number of bone marrow derived-cells followingtreatment with immunosuppressive agents.

[0035] Still another object of the present invention is to provide acomposition that is minimally toxic to the recipient.

[0036] These and other objects, features and advantages of the presentinvention will become apparent after a review of the following detaileddescription of the disclosed embodiments and the appended claims.

DETAILED DESCRIPTION OF THE INVENTION

[0037] The present invention may be understood more readily by referenceto the following detailed description of specific embodiments includedherein.

[0038] The present invention comprises a method comprisingadministration of a composition comprising a 3′-OH, 5′-OH, chemicallyunmodified, synthetic phosphodiester oligonucleotide sequences(hereinafter sequence) selected from the group consisting of SEQ ID NOs:1, 2, 3, or 4, and a pharmaceutically acceptable carrier, to an animalor a human in an amount effective to induce differentiation of cells.The compositions of the present invention may be used to treat diseaserelated to insufficient differentiation of cells. Such diseases includebut are not limited to leukemia, lymphoma, and non-malignant blooddisorders such as hemoglobinopathies, sickle cell disease andmyelodysplastic syndrome. The compositions of the present invention arebelieved to be useful for treatment of pancytopenia, anemia,thrombocytopenia and leukopenia. Other conditions that may be treatedwith the compositions of the present invention include lymphoma andnonmalignant blood disorders, including but not limited tohemoglobinopathies, sickle cell disease and myelodysplastic syndromes.The unexpected and surprising ability of these compositions to inducedifferentiation and to inhibit proliferation of leukemia cells addressesa long felt unfulfilled need in the medical arts and provides animportant benefit for animals and humans.

[0039] The present invention also comprises a method comprisingadministration of a composition comprising a 3′-OH, 5′-OH, chemicallyunmodified, synthetic phosphodiester oligonucleotide sequence(hereinafter sequence) selected from the group consisting of SEQ ID NO:1 (5′GTG3′), SEQ ID NO: 2 (5′GGGTGG3′), SEQ ID NO: 3 (5′GGGAGG3′) andSEQ ID NO: 4 (5′CCACCC3′) and a pharmaceutically acceptable carrier, toinduce maturation of progenitor cells in animals and humans. Such cellsinclude but are not limited to pluripotent stem cells, mycloid stemcells, lymphoid stem cells, immune cell precursors, and/or other cellsderived from these pluripotent stem cells, myeloid stem cells, lymphoidstem cells and immune cell precursors. The compositions of the presentinvention may also be administered to an animal or human to stimulateproduction and differentiation of cells from numerous sources, includingbut not limited to, bone marrow, liver, spleen, lymph nodes, thymus andcord blood.

[0040] As used herein, the word “sequence” refers a sequence comprisinga 3′-OH, 5′-OH chemically unmodified, synthetic phosphodiesternucleotide sequence selected from the group consisting of SEQ ID NO: 1(5′GTG3′), SEQ ID NO: 2 (5′GGGTGG3′), SEQ ID NO: 3 (5′GGGAGG3′) and SEQID NO: 4 (5′CCACCC3′).

[0041] The word “response”, as used herein refers to one or more of thefollowing non-limiting examples of responses: induced differentiation ofpluripotent stem cells, myeloid stem cells, lymphoid stem cells, immunecell precursors, and/or other cells derived from these pluripotent stemcells, myeloid stem cells, lymphoid stem cells and immune cellprecursors; induced differentiation of erythrocyte-like cells,monocyte-like cells or megakaryocyte-like cells; inhibition of cellularproliferation due to the induction of terminal differentiation;induction of hemoglobin synthesis; and stimulation of hemoglobinsynthesis.

[0042] As used herein, the phrase “effective in responsive cells” refersto the ability of the compositions of the present invention to inducedifferentiation and/or inhibition of proliferation and/or synthesis ofhemoglobin.

[0043] As used herein, the phrases “therapeutic treatment”, “effectiveamount” and “amount effective to” refer to an amount of a sequenceeffective to induce differentiation of cells, to inhibit proliferationof cells or to stimulate production of pluripotent cells such as bonemarrow-derived cells.

[0044] The word “disease”, as used herein, relates to a conditionwherein bodily health is impaired.

[0045] As used herein, the phrase “chemotherapeutic” is any agentapproved by a regulatory agency of a country or a state government orlisted in the U.S. Pharmacopoeia or other generally recognizedpharmacopoeia for use to treat cancer in an animal or human. As usedherein, the phrase “chemotherapeutic” includes immunosuppressive agents.

[0046] Administration of an effective amount of the composition of thepresent invention to an animal or a human is a therapeutic treatmentthat prevents, treats or eliminates a disease including, but not limitedto, leukemia, pancytopenia, anemia, thrombocytopenia, leukopenia,lymphoma, and non-malignant blood disorders such as hemoglobinopathies,sickle cell disease or myelodysplastic syndrome. Types of leukemiainclude, but are not limited to APL, AML, CPL and CML. Administration ofan effective amount of the composition of the present invention to ananimal or a human is also a therapeutic treatment that stimulatesproduction of progenitor cells, including but not limited to pluripotentstem cells, myeloid stem cells, lymphoid stem cells, immune cellprecursors, and/or other cells derived from these pluripotent stemcells, myeloid stem cells, lymphoid stem cells and immune cellprecursors. In a preferred embodiment, the present invention provides amethod to stimulate production and differentiation of marrow derivedcells. The compositions of the present invention may also beadministered to an animal or human to stimulate production anddifferentiation of cells from numerous sources, including but notlimited to, bone marrow, liver, spleen, lymph nodes, thymus and cordblood.

[0047] The terms “pharmaceutically acceptable carrier” or“pharmaceutically acceptable vehicle” are used herein to mean, withoutlimitation, any liquid, solid or semi-solid, including, but not limitedto, water or saline, a gel, cream, salve, solvent, diluent, fluidointment base, ointment, paste, implant, liposome, micelle, giantmicelle, and the like, which is suitable for use in contact with livinganimal or human tissue without causing adverse physiological responses,and which does not interact with the other components of the compositionin a deleterious manner. Other pharmaceutically acceptable carriers orvehicles known to one of skill in the art may be employed to makecompositions for delivering the oligonucleotide sequences of the presentinvention.

[0048] The oligonucleotide sequences of the present invention may becombined with pharmaceutically acceptable carriers and administered ascompositions in vitro or in vivo. Forms of administration include, butare not limited to, injections, solutions, creams, gels, implants,pumps, ointments, emulsions, suspensions, microspheres, particles,microparticles, nanoparticles, liposomes, pastes, patches, tablets,transdermal delivery devices, sprays, aerosols, or other means familiarto one of ordinary skill in the art. Such pharmaceutically acceptablecarriers are commonly known to one of ordinary skill in the art.Pharmaceutical formulations of the present invention can be prepared byprocedures known in the art using well known and readily availableingredients. For example, the compounds can be formulated with commonexcipients, diluents, or carriers, and formed into tablets, capsules,suspensions, powders, and the like. Examples of excipients, diluents,and carriers that are suitable for such formulations include thefollowing: fillers and extenders (e.g., starch, sugars, mannitol, andsilicic derivatives); binding agents (e.g., carboxymethyl cellulose andother cellulose derivatives, alginates, gelatin, andpolyvinyl-pyrrolidone); moisturizing agents (e.g., glycerol);disintegrating agents (e.g., calcium carbonate and sodium bicarbonate);agents for retarding dissolution (e.g., paraffin); resorptionaccelerators (e.g., quaternary ammonium compounds); surface activeagents (e.g., cetyl alcohol, glycerol monostearate); adsorptive carriers(e.g., kaolin and bentonite); emulsifiers; preservatives; sweeteners;stabilizers; coloring agents; perfuming agents; flavoring agents;lubricants (e.g., talc, calcium and magnesium stearate); solid polyethylglycols; and mixtures thereof.

[0049] The formulations can be so constituted that they release theactive ingredient only or preferably in a particular location, possiblyover a period of time. Such combinations provide yet a further mechanismfor controlling release kinetics. The coatings, envelopes, andprotective matrices may be made, for example, from polymeric substancesor waxes.

[0050] Compositions comprising one or more sequences and apharmaceutically acceptable carrier are prepared by uniformly andintimately bringing into association the sequence and thepharmaceutically acceptable carrier. Pharmaceutically acceptablecarriers include liquid carriers, solid carriers or both. Liquidcarriers are aqueous carriers, non-aqueous carriers or both, andinclude, but are not limited to, aqueous suspensions, oil emulsions,water-in-oil emulsions, water-in-oil-in-water emulsions, site-specificemulsions, long-residence emulsions, sticky-emulsions, microemulsionsand nanoemulsions. Solid carriers are biological carriers, chemicalcarriers or both and include, but are not limited to, viral vectorsystems, particles, microparticles, nanoparticles, microspheres,nanospheres, minipumps, bacterial cell wall extracts and biodegradableor non-biodegradable natural or synthetic polymers that allow forsustained release of the oligonucleotide compositions. Emulsions,minipumps and polymers can be implanted in the vicinity of wheredelivery is required (Brem et al. J. Neurosurg. 74: 441, 1991). Methodsused to complex an oligonucleotide sequence(s) to a solid carrierinclude, but are not limited to, direct adsorption to the surface of thesolid carrier, covalent coupling to the surface of the solid carrier,either directly or via a linking moiety, and covalent coupling to thepolymer used to make the solid carrier. Optionally, a sequence(s) can bestabilized by the addition of non-ionic or ionic polymers such aspolyoxyethylenesorbitan monooleates (TWEENs) or hyaluronic acid.

[0051] Preferred aqueous carriers include, but are not limited to,water, saline and pharmaceutically acceptable buffers. Preferrednon-aqueous carriers include, but are not limited to, a mineral oil or aneutral oil including, but not limited to, a diglyceride, atriglyceride, a phospholipid, a lipid, an oil and mixtures thereof,wherein the oil contains an appropriate mix of polyunsaturated andsaturated fatty acids. Examples include, but are not limited to, soybeanoil, canola oil, palm oil, olive oil and myglyol, wherein the fattyacids can be saturated or unsaturated. Optionally, excipients may beincluded regardless of the pharmaceutically acceptable carrier used topresent the oligonucleotide compositions to cells. These excipientsinclude, but are not limited to, anti-oxidants, buffers, andbacteriostats, and may include suspending agents and thickening agents.

[0052] One or more sequences may be administered alone, or incombination with other therapeutic modalities including, but not limitedto, chemotherapeutic agents, differentiating agents, immunotherapeuticagents, antimicrobial agents, antiviral agents or in combination withradiation therapy. Differentiating agents include, but are not limitedto, hemin, butyric acid, 5-azacytidine, cytosine arabinoside,hydroxyurea, guanosine, guanine, retinoic acid, trimidox,gamma-irradiation, mithramycin and chromomycin. Chemotherapeutic agentsinclude, but are not limited to, anti-metabolites, DNA damaging,microtubule destabilizing, microtubule stabilizing, actindepolymerizing, growth inhibiting, topoisomerase inhibiting, HMG-CoAinhibiting, purine inhibiting, pyrimidine inhibiting, metalloproteinaseinhibiting, CDK inhibiting, angiogenesis inhibiting, differentiationenhancing and immunotherapeutic agents. Dosages and methods ofadministration of these other therapeutic modalities are known to one ofordinary skill in the art.

[0053] Methods of in vivo administration of the compositions of thepresent invention, or of formulations comprising such compositions andother materials such as carriers of the present invention that areparticularly suitable for various forms include, but are not limited tothe following types of administration, oral (e.g. buccal or sublingual),anal, rectal, as a suppository, topical, parenteral, aerosol,inhalation, intrathecal, intraperitoneal, intravenous, intraarterial,transdermal, intradermal, subdermal, intramuscular, intrauterine,vaginal, into a body cavity, surgical administration at the location ofa tumor or internal injury, directly into tumors, into the lumen orparenchyma of an organ, and into bone marrow. Techniques useful in thevarious forms of administrations mentioned above include but are notlimited to, topical application, ingestion, surgical administration,injections, sprays, transdermal delivery devices, osmotic pumps,electrodepositing directly on a desired site, or other means familiar toone of ordinary skill in the art. Sites of application can be external,such as on the epidermis, or internal, for example a gastric ulcer, asurgical field, or elsewhere.

[0054] The compositions of the present invention can be applied in theform of creams, gels, solutions, suspensions, liposomes, particles, orother means known to one of skill in the art of formulation and deliveryof the compositions. Ultrafine particle sizes can be used for inhalationdelivery of therapeutics. Some examples of appropriate formulations forsubcutaneous administration include but are not limited to implants,depot, needles, capsules, and osmotic pumps. Some examples ofappropriate formulations for vaginal administration include but are notlimited to creams and rings. Some examples of appropriate formulationsfor oral administration include but are not limited to: pills, liquids,syrups, and suspensions. Some examples of appropriate formulations fortransdermal administration include but are not limited to gels, creams,pastes, patches, sprays, and gels. Some examples of appropriate deliverymechanisms for subcutaneous administration include but are not limitedto implants, depots, needles, capsules, and osmotic pumps. Formulationssuitable for parenteral administration include but are not limited toaqueous and non-aqueous sterile injection solutions which may containanti-oxidants, buffers, bacteriostats and solutes which render theformulation isotonic with the blood of the intended recipient, andaqueous and non-aqueous sterile suspensions which may include suspendingagents and thickening agents. Extemporaneous injection solutions andsuspensions may be prepared from sterile powders, granules and tabletscommonly used by one of ordinary skill in the art.

[0055] Embodiments in which the compositions of the invention arecombined with, for example, one or more pharmaceutically acceptablecarriers or excipients may conveniently be presented in unit dosage formand may be prepared by conventional pharmaceutical techniques. Suchtechniques include the step of bringing into association thecompositions containing the active ingredient and the pharmaceuticalcarrier(s) or excipient(s). In general, the formulations are prepared byuniformly and intimately bringing into association the active ingredientwith liquid carriers. Preferred unit dosage formulations are thosecontaining a dose or unit, or an appropriate fraction thereof, of theadministered ingredient. It should be understood that in addition to theingredients particularly mentioned above, formulations comprising thecompositions of the present invention may include other agents commonlyused by one of ordinary skill in the art.

[0056] The volume of administration will vary depending on the route ofadministration. Such volumes are known to one of ordinary skill in theart of administering compositions to animals or humans. Depending on theroute of administration, the volume per dose is preferably about 0.001to 100 ml per dose, more preferably about 0.01 to 50 ml per dose andmost preferably about 0.1 to 30 ml per dose. For example, intramuscularinjections may range in volume from about 0.1 ml to 1.0 ml. Theoligonucleotide compositions administered alone, or together with othertherapeutic agent(s), can be administered in a single dose treatment, inmultiple dose treatments, or continuously infused on a schedule and overa period of time appropriate to the disease being treated, the conditionof the recipient and the route of administration. Moreover, the othertherapeutic agent can be administered before, at the same time as, orafter administration of the oligonucleotide compositions.

[0057] Preferably, the amount of oligonucleotide compositionadministered per dose is from about 0.0001 to 100 mg/kg, more preferablyfrom about 0.001 to 10 mg/kg and most preferably from about 0.01 to 5mg/kg. In a preferred embodiment, the oligonucleotide compositions incombination with a chemotherapeutic agent is administered to an animalor human having leukemia in an amount effective to add to, synergizewith or potentiate the anti-neoplastic effect of the chemotherapeuticagent. Preferably, the amount of therapeutic agent administered per doseis from about 0.001 to 1000 mg/kg, more preferably from about 0.01 to500 mg/kg and most preferably from about 0.1 to 100 mg/kg. Theparticular sequence and the particular therapeutic agent administered,the amount per dose, the dose schedule and the route of administrationshould be decided by the practitioner using methods known to thoseskilled in the art and will depend on the type of disease, the severityof the disease, the location of the disease and other clinical factorssuch as the size, weight and physical condition of the recipient. Inaddition, in vitro assays may optionally be employed to help identifyoptimal ranges for sequence and for sequence plus therapeutic agentadministration.

[0058] The compositions of the present invention may also beadministered in vitro to affect differentiation of cells such aspluripotent stem cells, myeloid stem cells, lymphoid stem cells,progenitor cells, immune cell precursors, and/or other cells derivedfrom these pluripotent stem cells, mycloid stem cells, lymphoid stemcells, progenitor cells, and immune cell precursors.

[0059] The present invention is further illustrated by the followingexamples, which are not to be construed in any way as imposinglimitations upon the scope thereof. On the contrary, it is to be clearlyunderstood that resort may be had to various other embodiments,modifications, and equivalents thereof, which, after reading thedescription herein, may suggest themselves to those skilled in the artwithout departing from the spirit of the present invention.

EXAMPLE 1

[0060] Preparation of Sequences

[0061] Phosphodiester nucleotide sequences (SEQ ID NOs: 1, 2, 3 and 4)were prepared by Sigma-Genosys (Woodlands, Tex.) using Abacus SegmentedSynthesis Technology. Unless stated otherwise, the sequences weredispersed in autoclaved deionized water or in a pharmaceuticallyacceptable buffer such as, but not limited to, saline immediately priorto use.

EXAMPLE 2

[0062] Cells

[0063] The K562 cell line derived from the leukemic cells of a CMLpatient in blastic crisis is used as the standard model for determining,in vitro, the therapeutic potential of new differentiating compounds(Rutherford et al., Nature, 280:164, 1979; Drexler et al. DSMZ Catalogueof Human and Animal Cell Lines, 6^(th) ed., Braunschweig, Germany: DSMZ,1997). K562 cells were obtained from the American Type CultureCollection (ATCC, Rockville, Md.) and were cultured in the mediumrecommended by the ATCC.

EXAMPLE 3

[0064] Hemoglobin Synthesis by K562 Cells Cultured with SEQ ID NO: 1,SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4.

[0065] K562 cells were seeded in 1.0 ml at 2.0×10⁵ cells/ml in 6-wellflat-bottomed tissue culture plates for 72 hours with 100 μg of SEQ IDNO: 1, SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4. For hemoglobindetermination, the cells were washed twice by centrifugation inphosphate-buffered saline (PBS), stained with 0.2% benzidine(Sigma-Aldrich Canada, Oakville, Ontario) in 0.5 M acetic acid activatedwith 10% H₂O₂ (Gambari et al., Experimentia 41:673, 1985). After 10minutes incubation in the dark, the percentages of benzidine positivecells (hemoglobin positive cells) were determined by light microscopyusing an hemocytometer. Approximately 500 cells were counted for thedetermination of the percentages of benzidine positive cells. Cell sizewas also determined by light microscopy. Hemin (20 μg) was added to K562cells for 72 hours as a control for hemoglobin synthesis. Hemin wasobtained from Sigma-Aldrich Canada. TABLE 1 Evaluation of hemoglobinsynthesis (benzidine-positivity) and cell size of K562 cells SEQUENCE %of benzidine-positive cells Cell size None 5.4 normal SEQ ID NO: 1 17.4increased SEQ ID NO: 2 35.8 increased SEQ ID NO: 3 11.4 increased SEQ IDNO: 4 10.1 increased Hemin 17.8 normal

[0066] As shown in Table 1, SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 land SEQ ID NO: 4 induce the synthesis of hemoglobin by K562 cells and anincrease in their cell size, two measures of erythroid differentiation.

EXAMPLE 4

[0067] Upregulation of Rh D in K562 Cells Cultured with SEQ ID NO: 2 orSEQ ID NO: 3.

[0068] The Rh D antigen is the most important antigen of the Rh bloodgroup system. In humans, the Rh D antigen is expressed solely onerythrocytes (Cartron, Blood Rev. 6:199, 1994). K562 cells were seededin 1.0 ml at 2.0×10⁵ cells/ml in 6-well flat-bottomed tissue cultureplates for 72 hours with 2.5, 10.0, 25.0, 50.0 or 100.0 μg of SEQ ID NO:2 or SEQ ID NO: 3. The expression of Rh D at the cell surface wasmonitored by flow cytometry. After incubation, K562 cells were washedtwice by centrifugation with PBS and labeled with phycoerythrin(PE)-conjugated anti-Rh D monoclonal antibody (IBGRL research product,Bristol, Netherlands) for 30 min at 4° C. After washing twice withPBS-1% bovine serum albumin, cellular fluorescence was then determined.Flow cytometry was carried out on a FACSCalibur cell sorter (BectonDickinson, San Jose, Calif., USA) and analyzed using the programCELLQuest (Becton Dickinson). The fold increase in Rh D level overcontrol (0 μg oligonucleotide) was determined. Untreated K562 cells wereessentially negative for this marker. TABLE 2 Fold increase in Rh Dlevel over control in treated K562 with SEQ ID NO: 2 and SEQ ID NO: 3Concentration (μg/ml) SEQUENCE 2.5 10 25 50 100 SEQ ID NO: 2 1.7 x 2.5 x4.6 x 9.8 x 15.4 x SEQ ID NO: 3 2.0 x 2.2 x 2.9 x 6.5 x  4.7 x

EXAMPLE 5

[0069] Upregulation of CD41a Antigen in K562 Cells Cultured with SEQ IDNO: 2 or SEQ ID NO: 3.

[0070] The CD41a antigen, also named GpIIb/IIIa, is expressed onplatelets and megakaryocytes (Gruel et al., Blood 68:488, 1986). K562cells were seeded in 1.0 ml at 2.0×10⁵ cells/ml in 6-well flat-bottomedtissue culture plates for 72 hours with 2.5, 10.0, 25.0, 50.0 or 100.0μg of SEQ ID NO: 2 or SEQ ID NO: 3. The expression of CD41a at the cellsurface was monitored by flow cytometry. After incubation, K562 cellswere washed twice by centrifugation with PBS and labeled withphycoerythrin (PE)-conjugated anti-CD41a monoclonal antibody (BDPharmingen, Mississauga, Ontario, Canada) for 30 min at 4° C. Afterwashing twice with PBS-1% bovine serum albumin, cellular fluorescencewas then determined. Flow cytometry was carried out on a FACSCaliburcell sorter (Becton Dickinson) and analyzed using the program CELLQuest(Becton Dickinson). The fold increase in CD41a level over control (0 μgoligonucleotide) was determined. Untreated K562 cells were essentiallynegative for this marker. TABLE 3 Fold increase in CD41a level overcontrol in treated K562 cells with SEQ ID NO: 2 or SEQ ID NO: 3Concentration (μg/ml) SEQUENCE 2.5 10 25 50 100 SEQ ID NO: 2 3.0 x 3.9 x12.3 x 20.6 x 18.9 x SEQ ID NO: 3 3.2 x 3.7 x  8.3 x 13.4 x 11.7 x

EXAMPLE 6

[0071] Upregulation of CD14 in K562 Cells Cultured with SEQ ID NO: 2 orSEQ ID NO: 3.

[0072] The CD14 antigen is expressed at high levels on monocytes.Additionally, CD14 is expressed on interfollicular macrophages,reticular dendritic cells and some Langherans cells (Wright et al.,Science 249:1434, 1990). K562 cells were seeded in 1.0 ml at 2.0×10⁵cells/ml in 6-well flat-bottomed tissue culture plates for 72 hours with2.5, 10.0, 25.0, 50.0 or 100.0 μg of SEQ ID NO: 2 or SEQ ID NO: 3. Theexpression of CD14 at the cell surface was monitored by flow cytometry.After incubation, K562 cells were washed twice by centrifugation withPBS and labeled with fluorescein isothiocyanate (FITC)-conjugatedanti-CD14 monoclonal antibody (BD Pharmingen) for 30 min at 4° C. Afterwashing twice with PBS-1% bovine serum albumin, cellular fluorescencewas then determined. Flow cytometry was carried out on a FACSCaliburcell sorter (Becton Dickinson) and analyzed using the program CELLQuest(Becton Dickinson). The fold increase in CD14 level over control (0 μgoligonucleotide) was determined. Untreated K562 cells were essentiallynegative for this marker. TABLE 4 Fold increase in CD14 level overcontrol in K562 cells treated with SEQ ID NO: 2 or SEQ ID NO: 3Concentration (μg/ml) SEQUENCE 2.5 10 25 50 100 SEQ ID NO: 2 1.8 x 1.9 x3.9 x 7.2 x 9.8 x SEQ ID NO: 3 1.9 x 2.8 x 3.8 x 7.2 x 3.7 x

EXAMPLE 7

[0073] Induction of CD14⁺ Rh D⁺, CD14⁺ CD41a⁺ and Rh D⁺ CD41a⁺ Phenotypein K562 Cells Cultured with SEQ ID NO: 2.

[0074] K562 cells were seeded in 1.0 ml at 2.0×10⁵ cells/ml in 6-wellflat-bottomed tissue culture plates for 72 hours with 2.5, 10.0, 25.0,50.0 or 100.0 μg of SEQ ID NO: 2. The expression of CD14, CD41a and Rh Dat the cell surface was monitored by two dimensional flow cytometry.After incubation, K562 cells were washed twice by centrifugation withPBS and labeled with FITC-conjugated anti-CD14, PE-conjugated anti-CD41aand/or PE-conjugated anti-Rh D monoclonal antibody (BD Pharmingen) for30 min at 4° C. After washing twice with PBS-1% bovine serum albumin,cellular fluorescence was determined. Flow cytometry was carried out ona FACSCalibur cell sorter (Becton Dickinson) and analyzed using theprogram CELLQuest (Becton Dickinson). The fold increase in CD14⁺ Rh D⁺,CD14⁺ CD41a⁺ and Rh D⁺ CD41a⁺ level over control (0 μg oligonucleotide)was determined. Untreated K562 cells were essentially negative for thesemarkers. TABLE 5 Fold increase in CD14⁺Rh D⁺, CD14⁺CD41a⁺ and RhD⁺CD41a⁺ levels in K562 treated with SEQ ID NO: 2 Concentration (μg/ml)PHENOTYPE 2.5 10 25 50 100 CD14⁺Rh D⁺ 4.0 x 7.0 x 16.0 x 34.0 x 41.0 xCD14⁺CD41a⁺ 2.0 x 2.0 x  4.0 x 11.0 x 22.0 x Rh D⁺CD41a⁺ 20.0 x  22.0 x 45.0 x 87.0 x 100.7 x 

EXAMPLE 8

[0075] Induction of CD14⁺ Rh D⁺, CD4⁺ CD41a⁺ and Rh D⁺CD41a⁺ Phenotypein K562 Cells Cultured with SEQ ID NO: 3.

[0076] K562 cells were seeded in 1.0 ml at 2.0×10⁵ cells/ml in 6-wellflat-bottomed tissue culture plates for 72 hours with 2.5, 10.0, 25.0,50.0 or 100.0 μg of SEQ ID NO: 3. The expression of CD14, CD41a and Rh Dat the cell surface was monitored by two dimensional flow cytometry.After incubation, K562 cells were washed twice by centrifugation withPBS and labeled with FITC-conjugated anti-CD14, PE-conjugated anti-CD41aand/or PE-conjugated anti-Rh D monoclonal antibody (BD Pharmingen) for30 min at 4° C. After washing twice with PBS-1% bovine serum albumin,cellular fluorescence was then determined. Flow cytometry was carriedout on a FACSCalibur cell sorter (Becton Dickinson) and analyzed usingthe program CELLQuest (Becton Dickinson). The fold increase in CD14⁺ RhD⁺, CD14⁺ CD41a⁺ and Rh D⁺ CD41a⁺ level over control (0 μgoligonucleotide) was determined. Untreated K562 cells were essentiallynegative for these markers. TABLE 6 Fold increase in CD14⁺Rh D⁺,CD14⁺CD41a⁺ and Rh D⁺CD41a⁺ levels in K562 treated with SEQ ID NO: 3Concentration (μg/ml) PHENOTYPE 2.5 10 25 50 100 CD14⁺Rh D⁺ 7.0 x 8.0 x9.0 x 11.0 x 10.0 x CD14⁺CD41a⁺ 2.0 x 3.0 x 4.0 x  4.0 x  3.0 x RhD⁺CD41a⁺ 6.0 x 4.0 x 7.0 x 29.0 x 31.0 x

EXAMPLE 9

[0077] Inhibition of K562 Cell Growth by SEQ ID NO: 2 and SEQ ID NO: 3.

[0078] Terminal differentiation of K562 cells has been reported to stoptheir cellular growth (Bianchi et al., Biochem. Pharmacol. 60:31, 2000).K562 cells were seeded in 1.0 ml at 2.0×10⁵ cells/ml in 6-wellflat-bottomed tissue culture plates for 72 hours with 1.0, 10.0 or 100.0μg of SEQ D NO: 2 or SEQ ID NO: 3. Cells were counted after 24, 48 and72 hours of incubation using light microscopy and trypan blue dye. TABLE7 Number of K562 cells (× 10⁵) after treatment with SEQ ID NO: 2 or SEQID NO: 3 SEQ ID NO: 2 SEQ ID NO: 3 No 1.0 10.0 100.0 1.0 10.0 100.0Hours oligonucleotide μg/ml μg/ml μg/ml μg/ml μg/ml μg/ml 24 3.6 3.1 2.52.6 3.3 2.7 3.2 48 7.4 7.8 6.4 2.2 8.4 7.1 3.4 72 11.5  11.3  8.5 1.711.2  8.4 3.3

EXAMPLE 10

[0079] Differentiation of Human Committed Erythroid Precursor by SEQ IDNO: 2

[0080] Glycophorin A is the major glycoprotein of the human erythrocytemembrane. Maturation of committed human erythroid precursors ischaracterized by the expression of glycophorin A at the cell surface andby an increase in intracellular granulosity (Daniel and Greens, VoxSang. S2:149, 2000; Wheater et al., Functional Histology, a text andcolor atlas, 2^(nd) edition, Churchill Livingstone, U.K., 1987). Humancommitted erythroid precursors defined by the cell surface glycoproteinCD36 were isolated from expanded human cord blood CD34+ progenitors bypositive immunoselection of CD36+ cells (Clonetics, San Diego, Calif.,USA). Human committed erythroid cells were seeded in 1.0 ml at 1.5×10⁵cells/ml in 6-well flat-bottomed tissue culture plates for 96 hours with100.0 μg of SEQ ID NO: 2 or SEQ ID NO: 3. The expression of glycophorinA at the cell surface and the intracellular granulosity were monitoredby flow cytometry. After 48 and 96 hours of incubation, human committederythroid cells were washed twice by centrifugation with PBS and labeledwith PE-conjugated glycophorin A monoclonal antibody (CaltagLaboratories, Burlingame, Calif., USA) for 30 min at 4° C. After washingtwice with PBS-1% bovine serum albumin, cellular fluorescence wasdetermined. The intracellular granulosity was determined by the measureof side light scatter (SSC) using a flow cytometer. Flow cytometry wascarried out on a FACSCalibur (Becton Dickinson) and analyzed using theprogram CELLQuest (Becton Dickinson). The percentages of cells inSSC^(hi) glycophorin A⁺ and in SSC^(lo) glycophorin A⁺ were determined.SSC^(hi) is defined as >450 units; SSC^(lo) is defined as <450 units.TABLE 8 Percentages of human committed erythroid precursor cells inSSC^(hi) glycophorin A⁺ and in SSC^(lo) glycophorin A⁺ after treatmentwith SEQ ID NO: 2 or SEQ ID NO: 3 None SEQ ID NO: 2 SEQ ID NO: 3 48 h 96h 48 h 96 h 48 h 96 h SSC^(hi) glycophorinA⁺ 0.9 1.2 8.0 12.8 0.6 0.4SSC^(lo) glycophorinA⁺ 9.1 3.8 9.3 18.1 7.4 2.2

EXAMPLE 11

[0081] Effect of SEQ ID NO: 2 on Human Disseminated Chronic MyeloidLeukemia K562 Cells in Severe Combined Immunodeficiency Mice

[0082] Forty female severe combined immunodeficiency mice (SCID mice)were exposed to 1.8 Gy of radiation (rate: 7.5 Gy/h) from a γ source.Twenty-four hours after whole body irradiation (day 0), the 40 femaleSCID mice were weighed and randomized to form 4 groups (10 mice/group).The mean body weight of each group was not statistically different fromthe others (analysis of variance). Mice were injected intraperitoneally(ip) with 2.0×10⁷ K562 cells in 0.5 ml of RPMI-1640 medium. SEQ ID NO: 2(5′GGGTGG3′) resuspended in 0.9% sodium chloride USP was administratedip at 0.01, 0.1 and 1 mg per mouse per day from day 1 to day 29 (30days). A vehicle group was ip injected with vehicle (0.9% sodiumchloride USP) following the same schedule. The treatment schedule issummarized in the table below: TABLE 9 Dose/inj. Mice/ Route (mg/mouse/Vol./inj. Treatment Group Treatment group Admin. inj.) (ml) schedule 1Vehicle 10 ip 0 0.250 Q1DX30 2 SEQ ID 10 ip 0.01 0.250 Q1DX30 NO: 2 3SEQ ID 10 ip 0.1 0.250 Q1DX30 NO: 2 4 SEQ ID 10 ip 1 0.250 Q1DX30 NO: 2

[0083] The experiment was stopped at 120 days when mice were sacrificed.Survival was recorded two times per week.

[0084] The test evaluation expressed as a percentage (T/C %) and as theincreased life span value (ILS %) of the control evaluation wasdetermined. These are measures of the effectiveness of the compoundstested. Survival systems indicate a degree of success when T/Cpercentages exceed 125 and ILS percentages exceed 25. T is the mediansurvival times of animals treated with drugs and C is the mediansurvival time of control animals. T/C % and ILS % is expressed asfollowing:

ILS %=[(T−C)/C]×100

T/C %=[T/C]×100

[0085] Statistical analysis was performed using StatView200 (AbacusConcept, Berkeley, USA). Statistical analysis of the efficiency of thetreatment was performed using the Bonferroni/Dunn test (ANOVAcomparison). TABLE 10 Survival time of SCID mice having humandisseminated chronic myeloid K562 leukemia treated with SEQ ID NO: 2TREATMENT Group 2: Group 3: Group 4: Group 1: 0.01 mg 0.1 mg 1 mgvehicle SEQ ID NO: 2 SEQ ID NO: 2 SEQ ID NO: 2 Survival Survival timeSurvival time Survival time time (days) (days) (days) (days) 38 120  8549 54 57 34 120  46 54 57 41 54 75 75 57 34 120  34 57 38 120  31 99 34120  64 61 54 61 54 61 75 61 64 46 54 34 41 46 34 38 34 49 Mean ± 48.6 ±12.2 78.2 ± 34.9 52.1 ± 18.7 62.4 ± 24.6 sd median 46 61 41 49 ILS % —  32.6  −12.2   6.5 T/C % —  132.6   89.1  106.5

[0086] As shown in Table 10, SEQ ID NO: 2, at 0.01 mg/mouse/day,significantly increased the life span of SCID mice having humandisseminated chronic myeloid K562 leukemia (p<05). After 120 days, 4 of11 mice treated with SEQ ID NO:2 at 0.01 mg/mouse/day were alive whilenone of the 11 untreated mice was alive.

EXAMPLE 12

[0087] Effect of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO:4 on Differentiation of Bone Marrow-Derived Cells from Mice

[0088] The C57BL/6 mice are divided into 5 groups of 10 mice. Micereceive gamma-irradiation to induce a reduction in the number of bonemarrow derived-cells and bone marrow precursor cells. On day 0, group 1mice receive saline, group 2 receive SEQ ID NO: 1, group 3 receive SEQID NO: 2, group 4 receive SEQ ID NO: 3, group 5 receive SEQ ID NO: 4.Sequences resuspended in 0.9% sodium chloride USP are administrated ipat 0.01 mg per mouse per day for 7 days.

[0089] After 7 days of treatment, the mice are sacrificed. Cells presentin peripheral blood and in bone marrow are counted and their phenotypedetermined by flow cytometry. Hemoglobin levels are also determined.Mice in groups 2, 3, 4 and 5 have more bone marrow-derived cells thanthe mice in group 1. Mice in groups 2, 3, 4 and 5 have more mature bonemarrow-derived cells than the mice in group 1. The levels of hemoglobinare more elevated in mice in groups 2, 3, 4 than in mice in group 1.

EXAMPLE 13

[0090] Effect of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO:4 on CML in SCID Mice

[0091] K562 cells (2×10⁷ cells) are inoculated into SCID mice (severecombined immunodeficiency mice) as previously described (Beran et al.,Hematol. Pathol. 8:135, 1994). The mice are divided into 5 groups of 10mice. On day 0, group 1 mice receive saline, group 2 mice receive SEQ IDNO: 1, group 3 mice receive SEQ ID NO: 2, group 4 receive SEQ ID NO: 3,group 5 mice receive SEQ ID NO: 4. Sequences resuspended in 0.9% sodiumchloride USP are administrated ip at 0.01 mg per mouse per day for 30days.

[0092] After 30 days, the mice are sacrificed. Leukemic dissemination,leukemic cell phenotype and hemoglobin levels are analyzed. Mice inGroup 1 have the most leukemia cells and dissemination. Mice in groups2, 3, 4 and 5 have less leukemia cells and dissemination. Mice in groups2, 3, 4 and 5 show a higher number of differentiated K562 cells thanmice in group 1. Mice in groups 2, 3, 4 and 5 show more hemoglobinsynthesis than the mice in group 1.

[0093] All patents, publications and abstracts cited above areincorporated herein by reference in their entirety. It should beunderstood that the foregoing relates only to preferred embodiments ofthe present invention and that numerous modifications or alterations maybe made therein without departing from the spirit and the scope of thepresent invention as defined in the following claims.

We claim:
 1. A composition comprising a 3′-OH, 5′-OH, chemicallyunmodified, synthetic phosphodiester nucleotide sequence selected fromthe group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 and SEQID NO: 4, and a pharmaceutically acceptable carrier, wherein thecomposition is effective to induce differentiation of cells, to increasecells derived from pluripotent cells, or to treat disease associatedwith insufficient differentiation of cells when administered to thecells in vivo or in vitro.
 2. The composition of claim 1, wherein theinduction of differentiation of cells is induction of differentiation ofleukemia cells.
 3. The composition of claim 1, wherein the induction ofdifferentiation of cells is induction of differentiation of pluripotentstem cells, myeloid stem cells, lymphoid stem cells, progenitor cells,immune cell precursors, or cells derived from the pluripotent stemcells, the myeloid stem cells, the lymphoid stem cells, the progenitorcells, or the immune cell precursors.
 4. The composition of claim 1,wherein the disease is leukemia, lymphoma, a non-malignant blooddisorder, hemoglobinopathy, sickle cell disease, myelodysplasticsyndrome, pancytopenia, anemia, thrombocytopenia or leukopenia.
 5. Amethod comprising administration of an amount of a compositioncomprising a 3′-OH, 5′-OH, chemically unmodified, syntheticphosphodiester nucleotide sequence selected from the group consisting ofSEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO: 4, and apharmaceutically acceptable carrier, to an animal or a human wherein theamount is effective to induce differentiation of cells in the animal orthe human.
 6. The method of claim 5, wherein the animal or the human hasa disease associated with insufficient differentiation of cells.
 7. Themethod of claim 6, wherein the disease is leukemia, lymphoma, anon-malignant blood disorder, hemoglobinopathy, sickle cell disease,myelodysplastic syndrome, pancytopenia, anemia, thrombocytopenia orleukopenia.
 8. The method of claim 6, wherein the disease is leukemia.9. The method of claim 5, wherein induction of differentiation of cellsis induction of erythrocyte-like phenotype, monocyte-like phenotype,megakaryocyte-like phenotype, inhibition of proliferation or inductionof hemoglobin synthesis in cells.
 10. A method comprising administrationof an amount of a composition comprising a 3′-OH, 5′-OH, chemicallyunmodified, synthetic phosphodiester nucleotide sequence selected fromthe group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 and SEQID NO: 4, and a pharmaceutically acceptable carrier, to an animal or ahuman, wherein the amount is effective to increase differentiation ofpluripotent cells in the animal or the human.
 11. The method of claim10, wherein the animal or the human has received chemotherapy orradiotherapy.
 12. The method of claim 4, further comprisingadministration of a therapeutic agent.
 13. The method of claim 12,wherein the therapeutic agent is a chemotherapeutic drug, animmunosuppressive agent, a differentiating agent, an immunotherapeuticagent, an antimicrobial agent, an antiviral agent, radiotherapy, or acombination thereof.
 14. The method of claim 10, wherein the pluripotentcells are derived from bone marrow, liver, spleen, lymph nodes, thymusor cord blood.
 15. The method of claim 10, wherein the pluripotent cellsare derived from bone marrow.
 16. A method comprising administration ofan amount of a composition comprising a 3′-OH, 5′-OH, chemicallyunmodified, synthetic phosphodiester nucleotide sequence selected fromthe group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, andSEQ ID NO: 4, and a pharmaceutically acceptable carrier, to an animal ora human having a disease associated with insufficient differentiation ofcells, wherein the amount is effective to induce differentiation ofcells in the animal or the human.
 17. The method of claim 16, whereinthe disease is leukemia and the amount is effective to treat theleukemia.